Summary The goal of the proposed research is to characterize structures and interactions of receptors residing in plasma membranes, in particular those of immune cells. Membrane bound receptors play important roles in cells but their structures and molecular functions have been difficult to reveal. Importantly, the T-cell plasma membrane contains the T-cell receptor of antigens and the associated CD3 components crucial for signaling. Furthermore, the membrane contains the CD4/8, CCR5 and CXCR4, co-receptors of HIV. There are many other T-cell transmembrane proteins with numerous activities, such as CD2, CD45 or Ca2+ channels. For a number of these systems structures of extracellular or intracellular portions have been characterized in various groups including ours. However, interactions of transmembrane segments have been difficult to access on a structural level due to the complexity of handling membranes. Exceptions include the G-protein coupled receptors CCR5 and CXCR4, or the ? homodimer of the TCR and a model of the TCR transmembrane arrangement based on biochemical experiments and model building. Structures and functional aspects of complexes of T-cell membrane proteins with other proteins residing in other membranes are even more difficult to study but will be tackled here. We recently made a break through designing covalently circularized nanodiscs (cNDs) of variable size, which allows assembly of stable membrane protein complexes in a well defined near-native environment and dramatically facilitates structural studies of integral membrane proteins. Moreover, this approach provides access to structural studies of proteins residing in different membranes. We show preliminary data from non-circularized and circularized nanodisc since the latter was established only recently. It is now fully available and will be applied to the systems described. We will apply this technology in two specific aims: Aim 1. Characterize the interaction of viral particles with their receptors in circularized nanodiscs. As a proof of concept we will use EM methods to reveal detail of poliovirus interaction with its receptor CD155 in cNDs. This approach will be extended for imaging the interaction of HIV pseudovirus and/or gp160 with its co-receptors and will reveal details of viral engagement and pore formation. Aim 2: Determine the structure of the TCR/CD3 complex in cNDs and characterize the interaction with pMHC and CD8. Receptors residing in different nanodiscs will be linked with DNA handles to allow formation of cND sandwiches that mimic interactions of receptors from different cells.